The long-term objective of this project is to provide the biomedical research community with a new device for high-throughput single-molecule fluorescence assays. The domains of applications of these methods range from basic research to high-throughput diagnostics and drug screening. The goal of this research proposal is to develop an integrated system for multiplexed, high-throughput single-molecule assays. This system will be based on a new type of single-photon counting capable mutichannel hybrid photodetector (HPD) recently developed by Hamamatsu Photonics Corporation. We will incorporate this detector into a multiple excitation/multiple detection spot confocal microscope and demonstrate its capabilities in actual single-molecule assays. We expect this detector to find applications not only in fundamental research applications using single-molecule methods, but also in the emerging fields of high-throughput single-molecule diagnostics and drug screening. The specific aims of this research proposal are: (1) to develop an electronic interface and data acquisition system (including software) for the multichannel hybrid detector developed by Hamamatsu. (2) to integrate this system into a multiplexed single-molecule fluorescence excitation/detection confocal microscope. (3) to demonstrate the capabilities of this system for multiplexed, high-throughput single- molecule assays, by performing multiplexed fluorescence correlation spectroscopy (FCS) measurements on model systems of biological relevance as well as multiplexed single-molecule FRET measurements using an alternate laser excitation (ALEX) scheme.